Targeting Amyloids with [18F]AV-45 for Medullary Thyroid Carcinoma Positron Emission Tomography/Computed Tomography Imaging: A Pilot Clinical Study

Medullary thyroid carcinoma (MTC) is a malignant neuroendocrine tumor with a high recurrence rate. Amyloid plaques formed from the misfolding of calcitonin are the key characteristics of MTC. Herein, we conducted a first-in-human pilot clinical study by applying a β-amyloid-specific radiotracer, [18F]AV-45, to positron emission tomography (PET)/computed tomography (CT) imaging of MTC. The presence of amyloid plaques in the tumor tissue sections from five MTC patients was confirmed by hematoxylin and eosin (H&E) and Congo Red staining. [18F]AV-45 selectively accumulated in the amyloid plaques in the continued tumor tissue sections with similar distribution patterns to the H&E and Congo Red staining. In addition, the [18F]AV-45 uptake can be largely blocked by its nonradioactive reference compound. The [18F]AV-45 accumulation in the thyroid, neck lymph nodes, and muscles in healthy human subjects is close to the background indicated by PET/CT imaging. In the comparison PET/CT imaging study of a recurrent MTC patient, 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) showed an elevated uptake by multiple neck lymph nodes. In contrast, only one of these neck lymph nodes had increased [18F]AV-45 uptake. Postoperative histopathological analysis confirmed the [18F]AV-45 PET-positive lymph node as MTC with amyloid deposition, while other [18F]FDG positive lymph nodes were free from MTC and amyloid plaques. Thus, [18F]AV-45 showed the promise for the clinical PET/CT imaging of MTC.


■ INTRODUCTION
Medullary thyroid carcinoma (MTC) is a type of malignant neuroendocrine tumor derived from parafollicular C cells in the thyroid gland. 1 Although it only accounts for around 1∼2% of thyroid cancers, MTC causes 13.4% of all thyroid-related deaths. 2 Nearly 50% of MTC patients develop recurrent lesions, and the prognosis is closely related to the MTC development stage. 3 American Joint Committee on Cancer (AJCC) stage I, II, and III MTC patients have 10-year survival rates of 100, 93, and 71%, respectively, but the rate drastically decreased to 21% in AJCC stage IV MTC patients. Therefore, early diagnosis is vital for the survival of MTC patients. 3 Currently, the invasive fine-needle aspiration cytology (FNAC) is still the gold standard of MTC diagnosis, with a detection rate of about 50%. 4 Conventional anatomic imaging techniques, such as neck ultrasonography (US), contrastenhanced computed tomography (CT), and magnetic resonance imaging (MRI), are used to detect MTC metastatic lesions. 5 However, it is challenging for these morphological imaging modalities to visualize lymph node lesions and small liver metastasis. 5 Several single-photon emission computed tomography (SPECT) tracers, such as [ 99m Tc](V)-dimercaptosuccinic acid (DMSA), [ 123 I]-I-meta-iodobenzylguanidine (MIBG), and [ 111 In]-pentetreotide, have been used to image recurrent MTC but failed to provide sufficient sensitivity to localize metastatic lesions. 6,7 Positron emission tomography (PET) used for measuring cancer-associated biochemical variations is considered a superior functional imaging technique, offering a higher spatial resolution and better image quality of malignant lesions.  18 F]FDOPA measuring the increased activity of L-type amino-acid transporter 1 has the best diagnostic performance among these three PET tracers. 8,9 [ 18 F]FDOPA has been recommended by the European Association of Nuclear Medicine (EANM) as the first-line procedure for the diagnosis of MTC with PET, if available. 10 However, the availability of [ 18 F]FDOPA is limited to only a handful of PET centers worldwide because of its challenging radiosynthesis, prolonged preparation time, and low radiochemical yields. 11 Amyloid deposition is associated with many pathologically unrelated human diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, as well as MTC. 12,15 Although originated from the misfolding of different proteins, these unrelated amyloid plaques share a similar β-sheet structure. 13,15 MTC contains amyloid deposition formed from misfolded calcitonins that are rich in β-sheet structures. 15  Automated Preparation, Purification, Formulation, and Quality Control of [ 18 F]AV-45. In a Trasis Allinone automated synthesizer, fluoride-18 (∼40 GBq) was trapped on a QMA cartridge (preactivated with 10 mL of 1.0 M NaHCO 3 and 10 mL of sterilized water) and then eluted with a solution of Kryptofix222 (25 mg)/K 2 CO 3 (5 mg) in AcCN/H 2 O (4/1 v/v, 0.5 mL) to the reaction vial. Water was removed by two rounds of azeotropic distillation with anhydrous AcCN (2 × 0.5 mL) at 110°C. A solution of the precursor, AV-105 (1 mg) in anhydrous dimethyl sulfoxide (DMSO) (1.0 mL) was added to the reaction vial and heated at 110°C for 10 min. The reaction was cooled to 60°C before the addition of 3 M HCl (0.8 mL) for deprotection. The reaction was heated at 120°C for 5 min and cooled to room temperature (RT), to which 1 M NaOH (2.5 mL) and sodium ascorbate (6.5 mL, 5 mg/mL) were added to neutralize to pH 7.0. The crude reaction mixture was purified by a built-in liquid chromatography using a C18-HPLC column (Waters XBridge Prep, 5 μm, 4.6 × 150 mm). The mobile phase is AcCN/H 2 O (10: 9) containing sodium ascorbate (5 mg/mL) and NH 4 OAc (0.73 mg/mL) with a flow rate of 5 mL/min. The retention time of [ 18 F]AV-45 is 6.5 min. The [ 18 F]AV-45 was diluted with saline (20 mL), trapped on a tC18 cartridge, and released with ethanol (1 mL) into a collection vial containing saline (10 mL) with sodium ascorbate (5 mg/mL). The solution was filtered using a 0.22 μm Millipore sterile filter into a sterile vial for injection. The chemical and radiochemical purity and the molar activity of [ 18 F]AV-45 were determined using an Agilent 1200 HPLC with a Raytest GABI Star radioactivity detector, a diode array ultraviolet (UV) detector, and a ZORBAX Eclipse HPLC column (XDB-C18, 4.6 × 150 mm, 5 μm) with a mobile phase of AcCN/H 2 O (11: 9) and a flow rate of 1.0 mL/min. Kryptofix was determined using the kryptofix spot test with silica-based thin-layer chromatography (TLC). The discoloration during TLC was compared with a kryptofix reference solution (25 mg/mL). Radionuclide purity was determined using a germanium detector. Radionuclide identity is determined for the gamma spectrum emitted by the [ 18 F]AV-45. The half-life of the radionuclide was measured by the radioactive decay of [ 18 F]AV-45 over time. The solvent residual was determined by gas chromatography. Sterility tests were performed by adding the decayed [ 18 F]AV-45 to tryptic soy broth (TBS) medium (Soyabean casein digest) for 2 weeks at 25°C. Bacterial endotoxin was determined using endotoxin assay kits (Genescript).
Hematoxylin and Eosin Staining. The dewaxed and hydrated human MTC tissue sections were incubated with alum hematoxylin solution at RT for 5 min before sequentially rinsing with distilled water for 1 min, 1% HCl in ethanol for 20 s, and distilled water for 1 min. The sections were then incubated with 0.5% eosin at RT for 2 min, rinsed with distilled water for 1 min, dehydrated, and mounted with glycerol before observation under a Nikon Eclipse E600 microscope with a Nikon DXM 1200 digital camera (Nikon, JP).
Congo Red Staining. The dewaxed and hydrated human MTC tissue sections were incubated with alum hematoxylin solution at RT for 2 min and then in 0.5% HCl in ethanol for 20 s before rinsing with distilled water twice for 5 min. The sections were then incubated in 1% Congo Red solution at RT for 25 min, rinsed with distilled water for 2 min, dehydrated, and mounted with glycerol before observation under a Nikon Eclipse E600 microscope with a Nikon DXM 1200 digital camera (Nikon, JP).
[ 18 F]AV-45 Autoradiography and Blocking Study. The dewaxed and hydrated human MTC tissue sections were incubated in [ 18 F]AV-45 PBS solution (0.37 MBq/mL) with or without its nonradioactive reference compound (125 μg/ mL) for 40 min at room temperature. The sections were then washed with distilled water and air-dried prior to exposure to a multisensitive phosphor screen (PerkinElmer AQ5) at RT for 1 h. The phosphor screen was then scanned in a GE Amersham Typhoon Biomolecular Imager at a resolution of 25 μm. The images were analyzed with ImageQuant TL 8.1 software.

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Article [ 18 F]AV-45 PET/CT Imaging of Healthy Human Subjects. Four healthy volunteers (42−65 year old) free from thyroid diseases were intravenously injected [ 18 F]AV-45 (∼300 MBq) and rested for 1 h before PET/CT scan. For the anatomic correlation and attenuation correction of PET images, a low-dose CT (120 kV, 35∼170 mAs) was acquired from the vertex of the skull to the proximal femora. Subsequently, a PET scan of the same area was acquired with 2 min per bed position over seven bed positions.   To confirm the presence of amyloids in MTC, continued tumor sections from five MTC patients excised by surgery were stained with Congo Red and hematoxylin and eosin (H&E). Healthy human thyroid tissue sections were also stained accordingly as the negative control. Amyloid plaques were detected in all five tumor specimens as brown clusters by Congo Red and as pink clusters by H&E under a bright-field microscope indicated by red arrows (Figure 1a,b and c,d). No amyloid deposition was observed in the healthy human thyroid tissue sections (Figure 1g,h and i,j). Next, we investigated the selectivity and specificity of [ 18 F]AV-45 to bind to the MTC amyloid plaques in the continued tumor sections from the same five MTC patients by autoradiography. Continued healthy human thyroid tissue sections from the same specimens were also incubated with

■ DISCUSSION
Despite its rarity, MTC is the most fatal thyroid cancer with a high recurring rate. 2 The early diagnosis of MTC can significantly improve patients' prognosis. 3 Recently, [ 18 F]FDOPA has shown superior sensitivity and selectivity toward recurrent MTC. 10 However, the challenging radiochemical preparation of [ 18 F]FDOPA severely limited its clinical availability. 11 Thus, an alternative PET tracer that can be readily prepared on a standard automated synthesizer is required. Amyloid deposition has been recognized as a characteristic pathology of MTC. The main composition of MTC amyloid is formed from the misfolding of calcitonin that contains β-sheets in its secondary structure, 15